This project proposes to develop small-molecular inhibitors against the lethal factor (LF) of anthrax. In previous studies, we have developed two different types of LF inhibitors. Inhibitor DR9LF-1 is a peptide that Dinds to the active site of LF with high potency. The other group, represented by compound 1, is moderately potent and binds outside the active site. The present project is designed to develop compound 1 into a potent drug candidate and improve the stability of DR9LF-1 for the studies of synergistic inhibition of these two types of inhibitors in cells. The Specific Aims are: Aim 1. To improve potency and drug-like properties of compound 1 by structure-based design cycles. We plan to determine the crystal structure of LF-compound 1 complex and determine the essential group in compound 1 for inhibition. Such information will be utilized to design improved LF inhibitors. The repeat of the designing cycles, couple with assays for potency, selectivity, protection of cellular lethality and protection of animal from LF caused death, will acquire better drug properties in the inhibitors. At an advanced stage, the lead inhibitors will be tested for preclinical drug properties. Aim 2. To synthesize and study a stable peptide inhibitor, DR9LF-2, and use it in the study of synergistic inhibition vs. LF. New peptide inhibitor DR9LF-2 is designed to be stable but still provide a C-terminal group to chelate Zn++ in the active site of LF. After the synthesis and testing of this inhibitor for potency, DR9LF-2 will be used with new generations of inhibitors developed in Aim 1 for the study of synergistic inhibition of LF activities in cells and animals.